Preparation of ristocetin a salts



June 27, 1961 Filed Feb. 11, 1957 J. E. PHILIP ETAL PREPARATION OFRISTOCETIN A SALTS 2 Sheets-Sheet 1 RISTOCETIN "A" Inventors Julian E.Philip Jag. R. Schenck aq'ttom g June 27, 1961 Filed Feb. 11, 195'? WAVE NUMBERS, (.777.

J. E. PHILIP ET AL PREPARATION OF RISTOCETIN A SALTS 2 Sheets-Sheet 2 W41/5 LENGTH,

Mw p- M United States Patent-- 2,990,329 9 PREPARATION OF RISTO'CETIN ASALTS Julian'E. Philip, Lake Forest, and Jay R. Schenck,Waukegan',.lll., assignorsto Abbott Laboratories, Chicago,

" 111., aycorporationof Illinois Filed Feb. 11, 1957, Ser. No. 639,505 3Claims. (Cl. 167-65) This invention relates to a novel crystallineantibiotic substance and more particularly to a new crystallineristocetin A antibiotic and to the method of preparation thereof.

It has heretofore been found that the microorganism Nocardia lurida NRRL2430 produces during its growth in deep culture fermentation media asubstance designated generically as ristocetin which is a usefultherapeutic agent having properties not possessed by other previouslydescribed antibiotic agents. In the ristocetin fermentation media, twovery closely related ristocetins are produced and which have beendesignated as ristocetinA, and ristocetin B, each of which can beisolated in a distinct crystalline form. A method of fermenting theabove antibiotics is disclosed in the co-pending application for UnitedStates Letters Patent by Goldstein et al., Serial No. 373,692, filedAugust 11, 1953.

It is an object of the present invention to provide ristocetin A in acrystalline form substantially free of impurities and a method ofproducing same.

Other objects of the present invention will beapparent from the detaileddescription and claims to follow.

The foregoing objects of the present invention are readily achieved bypreferably aerobically growing in a deep culture fermentation media themicroorganism Nocardia lurr'da 'NRRL 2430 or otherristocetin-A-producing strain of the said Nocardia lurida organism whichare obtained by routine isolation and strain modification methodsincluding selection of culture organism, and exposure of the organism tomodifying means, such as X- ray, ultraviolet light, and chemical agents,and thereafter recovering the said ristocetin A as a solid crystallinematerial in the herein described manner.

The microorganism Nocardia lurida NRRL 2430, used herein for producingristocetin A, was isolated from a soil sample collected from the Gardenof Gods, Colorado Springs, Colorado. The isolation was carried out bystandard dilution procedures employing a solid medium, BaltimoreBiological Laboratories Trypticase Soy Agar, in Petri dishes.Structurally and functionally, this organism is found in the soil as amember of Nocardia genus according to the classification in BergeysManual of Deter ninative Bacteriology (6th edition).

A careful study of the morphology and physiology of the species N.lurida NRRL 2430 shows it to be distinctly different from any knownspecies of microorganism. Accordingly to the scheme of identification ofthe Nocardia species in Bergeys Manual of Determinative Bacteriology(6th edition), the Nocardia species are divided into two general groups.Group I is described as partially acid- 2,999,929 Patented June 27, 19611 known species of Nocardia by the several physical, cultural, andphysiological characteristics set forth as fol a m l When grown on theUpjohn Tryptone 'Dextrin Agar having the following composition: F

- Percent Tryptone 0 .5 Dextrin 1.0 Agar 2.0 Tap water, q.s. 100.0

moderate growth is observed with the Nocardia colonies having a 1 nim.diameter after four days growth, 2 mm. diameter after seven days growth,and 3 mm. diameter after eleven days growth. The colonies formed are circular in configuration becoming slightly irregular, are shiny with aglassy appearance becoming heavily crinkled and honey gold or light goldcolored (2ic), bamboo or chamois colored (2gc), substratal mycelium,cream white aerial mycelium forming around the edges of the colony, withscant cream aerial mycelium in the more heavily growing areas. The colorcode references used herein are in accordance with Color Harmony Manual(3rd edition), Jacobsen, Robert; Granville, Walter C.; Foss, Carl E.;1948, Container Corporation of America.

When examined microscopically the mycelium of N0- cardia lurida NRRL2430 is observed to grow moderately fast organisms with stronglyrefractive cells; non-prois not diastatic and therefore isdistinguishable from the I group H species. None of the 33 Nocardiaspecies described in Bergeys Manual has the family of characteristicscorresponding to those of Nocardia lurida.

The Nocardia lurida species NRRL 2430 used in the dense and has amonopodial type branching structure; Early in growth it is non-septate.However, segmentation of the mycelium begins in 48 hours and completefragmentation has taken place in 72 to 96 hours. The fragments oroidio-spores are rod-shaped with well-dc fined square ends. They measureapproximately 0.8 to 1.5 microns in length. The mycelium measuresapproximately 0.5 micron in width. Single loop and double loop coils areobserved in the mycelium as the culture ages when grown for microscopicobservation. Conidia are not observed. The organism is not acid-fastwhen stained by conventional techniques. I The ability of themicroorganism Nocardia luridd NRRL 2430 producing the antibiotic of thepresent invention to utilize various sources of carbon indicated in'thefollowing table:

TABLE I Utilization of carbon Carbon Source Utiliza- Rate of I tionGrowth Xylose Moderate Pentmns Arahinnsa D Do. Hexoses-. D0. Do.Ketoses; i

Di-Saccharldes...

Ra ld. Tri-Saccharlde--. Poly-Saccharlde.. I Glucoside Slow.

Moderate. Rapid. Almhn'l': I

I- Slim. Do. Acids Sodium Si'mninatn Do.

1 Sodium Acetate 1 Moderate.-,- Sodium Potassium Tartr te.Hydrocarbon-.- Paraflin Do.

Control (no carbon source added).

, 3 It should be understood that the above tests for utilization ofenergy sources are carried out under special assessagrowth conditionsand that the failure of the antino- 1 mycete to utilize certain energysources under the test;

'4 wherein strips are suspended in the developing (irrigating) solventcontained at the bottom of the cylinder, preferably after equilibratingthe strips with the atmosphere of the cylinder. The stripsrwere allowedto remain in the cylinder and developv for about 16. hours, at atemperatureof about 28 -C. and thereafter dried andgde veloped in theusual manner. The test organismio'i; itli l bidautographs was Bacillussubtit lis ATCC 10707 and the paper employedwas a standardstriprofEaton-Dikeman #613. The solvent systems found particularly useful indifi erentiating the antibiotic ristocetin B from all other known,antibiotics are: n-butanol saturated with deionized water, n-butanol;saturated-with deionized-Water andhav 1 times l as u a s hq s lu filie wr alr crq and, i q d 9 as stab e n ard; 3- On ater er t c r h ra ou cSUPPl l welrmmml prod c smola o y p qr h ike. The preferred nitrogensources include a wide variety of substances of both animal andvegetable origin, such as soybeanrneal, meat extracts, corn steepliquor, distillers. sol hles and other sources. including casein, aminoacid. p pm s nd h l lnorsanicr ogen such as.nitra tes, nitrate salts orammonium salts also beernployed.

The nutrient inorg 'c salts incorporated in the medium include the.customary. salts capable of yielding ions of i tsa 'e s ls um z h e n h1 iii sulfate, and the like. i r l'. As is necessary for the. growth anddevelopment of other microorganisms, essential trace elements should beincluded in the culture medium. Suchtrace ele-..

mentsare. suppliedlas impurities. incidentalto the additionv of'theother constituents of the medium.

ing 2 w./-v. paratoluene sulfonic acid addedtonbutanol rich layer, n-butanol saturated with deionized water having 2.0% w. /v. paratoluenesulfonic acid and 2.0% piperidine w./v. added to the butanol rich layer,

' methylisobutyl ketone saturated with deionized water, and

methanol 80parts and deionized water 20 parts v./v. containing 1.5sodium chloride w./v. and having the strips buifered'with a solutioncontaining .95 M sodium sulfate and .05 M sodium acid; sulfate (Nal-ISOQIt has been found that ristocetin B moves less rapidly than ristoceu'n Ain the latter solvent system, and where there is a mixture of;ristocetin B and ristocetin A, two clear longitudinally spaced zonesappear in the bioautograph. Comparison of the position of the zone willestablish which antibiotic is present. Under optimum conditions using afresh solvent mixture consisting ofthe said 80 parts methanol and 20parts water containing the specified. salts, the Rf value of 'ristocetinB is about .16. and the Rf;

value-of ristocetin A isabout .40.

"As" heretofore specified, the preferred method of producing ristocetinA is, by deepculture aerobic fermentatio'n."v However, for thepreparation of limitedamounts of ri tocetin A, shake 'flask andv surfaceculture bottles can be, employed. In, accordance with the usualprocedure, it isdesirable to first grow. avegetative inoculum of the N ciaur d mgani m andi l a s e QtiYevtat ive aseptically to. the.fermentation tanks. T e. med um n which the e et tive nw l m s. a e:duced,'can be the same or different-from that to p'roi duce theantibiotic.

The Nocardia lurida strain 'NltRL 2,430 is grown in the fermentationprocess at a temperature of between about 22 C. and 28 Themaximum=yields of ristocetin A. arepresentlyobtained. within. about.four: daysof fer:

' 'men tation although appreciable yields are produced evenafterprolonged periods of fermentation. The rate ofproduction ofristocetin A in the fermenta-- tionrnedia is conveniently determined bythe paper disc assay methodidescribed by 11.1111. Loolet al;, J:Bacteriol ogy 50, 701 (1955). In-assaying the fermentation media of thepresentl inventiom'the clarified solution'is placed upon the paper discand the assay proceeds in the same manner as describedjin theabovefreference. It has been found desirable, however, inpreparing theassay standard; and th e assayzsamplesjtojmploy a somewhat moreconcentrated solution ofthe antibiotic. Thus, the standard is preferablydiluted inpII6l0 phosphate bulfer r(l%)-to lilqrncg /ml j fw mcgrlmL, 20mcg ./ml., and-l0 meg/ml.- Aftcntheassay samplfsiarejplated, the platesare incubated overnight at 30 Cw Thereafter, zone sizes are readinmillimetersrid the, standard slope determined in the usual manner. Q

Therpresence of ristocetin A and; any ristocetin B. can be. readilydetermined-by paper; chromatographic procedures in accordance-withtheusualascending strip. technique The solvent used iri accordancewithfthe herein d6: scribed standard technique was a. freshly preparedsolutionof'methanol parts and deionized water 20 containing 1.5% sodiumchloride w./v. 'with the strips,

bufiered'with a solution containing .95 molarsodiumsul fate and .05molar sodium acid sulfate. Theareas indig. catedin. black are zoneswhere no growthof the bacterial test organism B. subtilis occurred.

5 The antibiotic ristocetin A produced by fermentation of aculture ofNocardia lurida NRRL 2430 is recovered from the fermentation medium, byutilizing the adsorptive technique. Thus, the antibiotic ristocetin A isadsorbable from the clear solution thereof upon various surfaceactive'niaterials, such as carbon, ion exchange resins and; cellulose;Useful results-are produced by adsorbing the antibiotic on activatedcarbon, such as Darco-G-60, and on other common commercial carbons. Theclear; fil trate, after the separation of adsorptive material, may be,discardedand the carbonis washed with water torernove impurities; Theadsorbed antibiotic activity free of. the fennentation. beer is then;eluted batchwise by. slurrying with an aqueoussolution ofa solventselected from the group acetone; methanol; ethanol, isopropanol,tertiarybutanol; andisimilar organic polar solvents, saidsolutionbeingacidified-with a dilute acid, such as sulfuric acidor aceticacid.'I-he-slurrying operation is prefer-ably repeat: ed-several timesand thefinal volumeis approximately one-third that of the. starting beer. Theaqueousacetone' eluateisconcentrated;to remove the acetone and thepH ofthe-resulting aqueous solution is adjusted to between about. pH 4.0 and.5.0. The potency of the ristocetin prior. to the; subsequent;chromatography. generally a be: tween-abqut-60and mcg./mg. v I

Carbon chromatographic purification .andsepfiration of ristocetin-A fromother antibiotics is preferably-efiected by passing. an aqueous solutionof the ristocetin Apre; pared in-the above manner and-having-a pfl ofbetween PH 4.0. and 5.0,.through a column comprised of activated w ensuc 5 Demo containing ams ar qn OeIite 545', to improve the new rate.The activityis adorbed on the column and the, column is washed withwater to remove extraneous, water-soluble unadso'rbed,

material. Development of the activity through the column is thenaccomplished by the flow through the column ing solution is aboutl6%acetone. The presence of ristocetin A is detected by paper stripchromatography (FIG. 2) and those solutions having an R in the rangebetween about .4.6 under the conditions hereinafter to be describedwhich specify using a freshly prepared develop ment solution arecombined and concentrated to give an antibiotic having a potency ofabout 600 units/mg.

Ristocetin A can be further purified by passing the latter aqueoussolution of the said antibiotic having a potency of about 600-800units/mg. through an acid-washed alumina column (pH 3.0 with H 50 Fiftymilliliters of alumina in methanol was used per gram of total solids inthe ristocetin preparation. The alumina column to which is applied theconcentrated aqueous solution of ristocetin A (50% w./v.) is developedby first washing with a substantially water-free polar solvent, such asabsolute methanol, followed immediately by eluting the ristocetin Aactivity with a polar solvent-water solution, such as a 50:50water-methanol solution. The bulk of the ristocetin activity is obtainedin the said solvent-water fractions which on concentrating produces aristocetin product having a potency of about 800l200 units/mg.

The amorphous ristocetin A product obtained from the alumina column bytreatment in the above manner is crystallized by dissolving a portionthereof in a wateralcohol solution, such as a water-ethanol solution,and centrifuging to separate" an oil precipitate. Additional ethanol isadded to the aqueous alcohol solution and further oily precipitationoccurs which is removed by centrifuging. The saturated aqueous-alcoholsolution free of precipitated oil is allowed to stand at roomtemperature after scratching the tube until crystallization is com.-plete. a

The following specific examples areset forth solely for the purpose ofillustrating the present invention and should not be construed to limitthe invention to the precise ingredients and proportions specified.

EXAMPLE I An agar slant of oatmeal agar is prepared by steaming 30 g. ofrolled oats for 30 minutes in one liter of tap water after which thematerial is filtered through cheesecloth and 2% agar added to form asolid medium and is thereafter inoculated with a soil stock sample ofthe microorganism N ocardia lurida NRRL 2430 described by the co-pendingapplication Serial No. 373,692, filed August 11, 1953. The slant isincubated at 28 C. for between four and seven days. Sterile distilledwater is added to the slant and the surface growth removed by scrapingwith a sterile inoculating loop.

Standard 500 ml. Erlenmeyer flasks containing 125 ml.

7 of the following medium:

are sterilized at 121 C. for 30 minutes under steam pressure'and areinoculated with the surface growth from the above sterile agarslant. Theflasks arefincubate'd f on a rotary shaker machine operating at 240-r.p'.-m. 2.25 3 inch accentricradius at a temperatureof 28 Cyfor 'aperiod of 48 hours. 4% by volume of the growth from the Erlenmeyerflasks is used to inoculate two additional flasks containing the samefermentation medium. 'lThe se newly inoculated flasks are incubated for48 hours at 28 C; under the same rotary conditions. At the end' of the48-hour growth period theinoculum is ready to be transferred to theproduction medium.

Thereafter, flasks containing the following growth medium: Cerelose gMolasses I g 20 Peptone g Tap water liter 1 are inoculated with 4% byvolume of the growth contained in the latter pair of Erlenmeyer flasksand are incubatedin the same manner as the above-mentioned Erlenmeyerflasks. After four days of incubation on the rotary shaker machine, thegrowth is harvested and the pooled beer is used as a source of the newantibiotic substance of the present invention. Assays are carried out bythe herein described paper disc assay method using Bacillus subtilis andwhich indicate on the average of at least about 40 units/ml. I

which has been steam-sterilized at 121 C. for one hour is inoculatedwith 300 ml. (25% by volume) of the ristocetin-B-producing culture grownin the shaken flasks The fermentation is carried out at 26 C. with anaeration rate of 0.8 liter vi in the manner described above.

of air per liter of medium per minute. Agitation is accomplished bymeans of a 4-bladed impeller, 8" in diameter, which operates at 480 rpm.Lard oil containing 2.5% octadecauol is used to control foaming. Thefermenters are harvested after four days and the beer when assayed bythe herein described paper disc assayv method shows yields of at leastabout units/ml.

EXAMPLE II The beer obtained by the fermentation process bra ample I isharvested by adding to the whole be ,;2%., wt./unit volume of a filteraid, such as Celite 545, and

filtering the beer. The ristocetin in the clear beer is then adsorbed onactivated carbon by adding to the clarified beer 10 g. Darco G-60 perliter of beer filtered.

The ristocetin activity is eluted from the carbon with three portions of40% aqueous acetone containing 4 ml. of 6 N sulfuric acid per liter ofacetone solutiom, Th total volume of eluate used is about 300 ml.perliter of starting beer.

The aqueous acetone eluate is concentrated to remove the acetone and thepH of the aqueous ristocetin solution is adjusted to about pH 5.0 andthe clear ristocetin solution is introduced into a carbonchromatographic column. The potency of the ristocetin solution prior tothe chromatography is generally about 200 units/mg. as determined by theBacillus subtilis plate assay method.

The ristocetin aqueous solution is thus further purified by passingthrough a carbon chromatographic column comprising 6 g. of carbon and 6g. of a filter aid, such as Celite 545, per gram of solids having apotency of 1 200 units/mg. antibiotic activity. The said carbon col umnis preferably prepared by mixing the carbonand celite as a thick slurryin water containing a wetting agent, such as tergitol penetrant, pouringthe slurry v.rl L .-...v:.,

through the activated cari Y crystal habit and was biaxial negative. v

analysis thesaid crystals were found to contain 51.96% t solutioncontaining 700 grams total solids and having a potency of approximately200 units/mg. is applied to a carbon column having a'diameter of 6inches and containing 4350 grams of Darco G-60 and 3380 grams ofCelite'SAS.

. TABLE-n Fractions Volume Soln., Million Paper Strip (liter) PercentUnits Rt Acetone 95 -8 o 126 8 46.5 0.40 '54 16 38.3 0545 36 16 4.2 0 18and 0.45 18 32 4.5 0 17 and 0.44 18 32 15.7 0116 38 32 10.0 0.16

Fractions B and C containing rist'o'cetin A are combined and furtherpurified as in Example 111.

EXAMPLE III The "ris'foce'tin A product obtained by "the process ofniranipleul is. further purified y 'chrb'matbgraphing n aqueous solutionof "the s'aidatitibiotic through an acidwashed alumina column. Thealumina column is prepared' by slurrying alumina, such as Alorco alumina(grade F), in water and adding thereto sulfuric aciduntil-acon'stantpI-I of 3.0 is attained. The alumina thus treated is:washed with water to rerriove excess acid an is then' 'w'ashed withmethanol and air dried.

Ristoceti'n A amorphous (63.5 'g.) from the carbon column process ofExample II and having a potency of about SSUu/mg. isdissolved in 125 ml.of water. The solutio'nof the risto'c'etin- A thus prepared is appliedto a column'con taining 3.0 kg. of alumina acid washed in the above'n'lanner after the introduction of the said ristocetin solution intothe column, the column is washed with 2.0

liters of methanol and developed immediately by the addition of portionsof a :50 methanol-water solution. The {following is 'a summary of thefractions collected fromthe column:

1 Fractions volume, 1. Total Units, M.U.

A- 7.0 B. 2. O 0. 2 C 2. O O. 6 D 2. U I 0. 7 E 13. 7 13. F- 11. 0 9. G7. O 2. H a 7.0 o.

ime 25.8 M.U. in the above fractions E, F and G were combined andconcentrated to dryness to give 27.0 grams ofpurified product.

EXAMPLE IV l rom the above solution containing 2 .7.0 grams off fromExample III ayeld oil 1 0.78

ristocetin, a product I gram of'fine crystals was obtained which hadaprismatic On chemical c, 5.67% 3.24% N,'1.25% s, and 35188% o bydifference. 7 V 1' I EXAMPLE V Ristoc'etin A sulfate has also been,obtained in crystalline 'form b'y concentrating 14.0 liters 'of the"ristocetin A fractions from a carbon colurrii1(l'6% acetone'containing1 'ml.'/l. of '6NH2S04) r0200 ml. The "p11 wasted- .justed 106.4 withsolid Ba(OH) .8HO "with stirring and the filtered solution waslyophilized.

The 8.0 grams ofthe clry amorphous 'risto'c'eti'n A was dissolved in 30ml. 'warm'water (50-60 C.), centrifuged clear and'the solution'wasallowed'to cool toro'om temperature. Crystallization began in a fewminutes and was allowed to continue until completed. The crystals werethen washed with 3 ml. H 0 and air dried to give a yield of ristocetin Acrystals of 3.45 grams. Ihe potencyof the said crystals was 910-930u./r'ng. On chemicalanalysis, the crystals were found to comprise 51.88%C, 5.49% H, 4.61% N and 1.06% S.

A photomicrograph of the uni'agrial positive hexa onal prismatic rods ofristoce'tin A obtained in the above manner isshown in 'FIGURE 1 of thedrawing.

The X-ray ditfraction pattern of the latter 'r'i'stocetin A crystals hasbeen obtained by the use of nickel filtered Xf-raysfrom a copper target(CuKa,7\='l.5418 A.) with (1) a GE. Ipowder camera of effective radius7.16 cm. which registers spacings up to 20 A. and (2) a 'G.E. fla't filmcamera with a specimen to film distance or l0 cm. which registerslong-spacings from 4 A. to 50 A. crystals of ristocetin A'wer'eintroduced iiit'oa glass capillary with a little mother liquor and thecapillary was sealed at each end. The d-spacings and estimated relativein tensities from these patterns are as follows:

Paper chromatography tests on a solution ofcrystalline i ristocetingAsulfate obtained in the foregoing manner were carried out in 1000 ml.graduate cylinders by the ascending -strip technique wherein strips weresuspended inthe developing (irrigating) solvent contained at the bottomof the cylinder by frictionally engaging the strip between the groundglass cylinder stopper and the'neck portion of the cylinder. In some ofthe solvent systems employed, the strips were equilibrated with theatmosphere of the cylinder by allowing them to hang above the solventsurface for a period before being lowered into the Cry's tallineristocetin A sulfate is produced from'the combined high potencyfractions E, F and G obtained in Example III by dissolvingthe 27.0 gramsof product in1 l08 "ml'.'o water and adding 216ml. of 95% ethanol ed bywarming until solution is substantially comstand; and erystaliizatienproceeded slowly over a pence of about one week.

The solution is centrifuged-clea and allowed to solvent. All of thesolvent systems were run in :an air incubator maintained at 28 C.immiscible solvent systerns were allowed to separate at 28 C. The testorganism for bioautographs was Bacillus subtilis'A'TCC l 19707 and thepaper employed was a standard strip of Eaton-Dikeman #613.

Solvent system I:'

n-Bu'tahbl saturated with deionized water Etiuilibrh'tidn-Ii hour'sDeveloping 'tifil'e'16 to 17mins Rf=0 n-Butanol saturated with deionizedwater. 2.0% w./v. paratoluene sulfonic acid'added to n-butanol richlayer Equilibration-3 hours Developing time-l6 to 17 hours Solventsystem III:

n-Butanol saturatedv with deionized water. 2.0% w./v. paratoluenesulfonicacid and 2.0% 'piperidine v./v. added to the butanol rich layerEquilibration-3 hours Developing time-16 to 17 hours Solvent system IV:

Methylisobutyl ketone saturated with deionized water Equilibration-noneDeveloping time-3 hours Solvent system V:

Methylisobutyl ketone saturated with water to which is added 2.0%paratoluene sulfonic acid w./v.

Equilibration-none Developing time-. 3 hours Solvent system VI: I

Methylisobutyl ketone saturated with deionized water plus 2% v./v.piperidine Equilibration-none Developing time3 hours Rf== I Solventsystem VII:

Deionized water saturated with methylisobutyl ke-,

tone

Equilibration-none Developing time-3 hours Solvent system VIII:

Deionized water saturated with methylisobutyl ketone with l.0% w./v.paratoluene sulfonic acid added Equilibration-none Developingfime-Zihours Solvent system IX:

Deionized water saturated with methylisobutyl ketone to which is added1.0% v./v. piperidine Equilibration-none Developing time-3 hours Solventsystem X:

3 parts deionized wateril part of. a mixture of methanolzacetone 3:1v./v.' Adjust pH to 10.5 with NH OH then back to pH 7.5 with phosphoricacid Equilibration-none Developing time-3 hours Solvent system )H: 1 v

Methanol 80 partszdeionized water 20 parts v./v. plus 1.5% NaCl w./v.added to the solution. Strips are buflered with a solutioncontainingr.95 M Na SO- and .05 M NaHSQg' Equilibration-3 hoursDeveloping time- 16 to 17 hours Solvent system AA:

Amyl acetate saturated with 0.1 M potassium phosphate buffer at 6.15.The buffer is made from K HPO and KH PO salts in deionized waterEquilibration-3 hours 7 p 1 Developing time-+16 to 17 hours (solventpar-t goes to neck of cylinder and evaporates) Solvent system II:

10 Solvent system MO: w 40 m1. n-butanol, 10 ml. methanol, 20 ml.deionized water. Mix together and add excess methyl orange(approximately 1.5 gms.) and let stand at 28 C. Separate fromundissolved methyl orange Equilibration'3 hours Developing time-46 to 17hours Rf=- y Ristocetin A on chemical analysis is found to consist ofthe following elements: carbon, hydrogen, nitrogen, and oxygen. Severalcrystalline ristocetin A sulfate samples when prepared in accordancewith the foregoing procedure, on chemical analysis were found to containC, H, N, O, and S in the range of 52-63% carbon, 5.55.7%- hydrogen,4.7-5.9 nitrogen, 34.137.0% oxygen, and 0.8-1.3 sulfur. The lattersulfate salt can be readily converted to the chloride salt by passing asolution of the said sulfate over an anion exchange resin, such asAmberlite IR-4B in the chloride form. Other salts of ristocetin can beprepared by passing a solution thereof through a resin column of theforegoing type which has been regenerated by treatment with theappropriate anion. The phosphate and picrate salts of ristocetin A havealso been prepared by precipitation from aqueous solution after additionof the acid thereto.

The infrared adsorption spectra of the crystalline sulfate of ristocetinA in a petroleum mineral oil (Nujol) mull, using a Perkins-Elmer model21 double beam infrared spectrometer, is shown in FIGURE 2 of thedrawing. a The Nujol mull of ristocetin A when adjusted to aconcentration at Whichthe broad adsorption hand at 9.4,u transmits 20%,exhibits the following adsorption bands, the infrared spectrum between2-15 Broad (S). 3 ,420 (Nujol). 6,06,, (M). 6 28 6.64 u (M) t 6.84;: il- 7,28 7.6-7.8, Broad (W). 8.25, 9.4," Broad (S). 12.0-12. Broad (W);13.9,u (N i S =strong.

M= medium.

. W =weak.

; The crystalline ristocetin A sulfate gives a strongly positive testfor carbohydrate with anthrone reagent and a strongly positive test forreducing sugars after dilute'acid hydrolysis. Y

The biuret, 'Sak-aguchi, and maltol tests on the crystalline ristocetinA sulfate were negative. The phospho molybdic acid color test of Folinand Ciocalteu for phenols Was positive for the ristocetin A and theninhydr'in tests were positive after acid hydrolysis.

6 Using a Tiselius electrophoresis apparatus, the electrophoreticmobility values 'for the crystalline ristocetin A sulfate are 4.4 |10'-cmfi/sec. volt at pH 3 (phthalate buffer, u=0.05) and 7.4 10- om. /sec.volt at pH 6.2 (phosphate bufier, u=0.05).

The isoelectric point of the crystalline ristocetin A sulfate is 8.1 fora glycine-sodium hydroxide bufler at an ionic strength of 0.05. i Thediffusion constant for the crystalline ristocetin A sulfate is 106x10cm'. /sec.

The ristocetin A sulfate crystals shown in FIGURE 2 of the drawing arebirefringent and exhibit an average refractive index of 1.575; w

The specific rotation of the crystalline sulfate of ristocetin A inwater was between and -l33.

Upon subjecting a crystalline ristocetin A sulfate (about 7 11 strips ofpaper (Whatman 0.3 mmJ'aboutfloiinehesin length iir'0134 M sodiumphosphateb pipetting about x611 a l in /cc; solfitionof crystalline iistocetin Aonto "the said strips followed "byspltttz'ing the stripsbetween thick" gtlass plat Withfthe'endsof the strips placed in thebuffer which ridged to a normal potassium chloridesolutionthrough' agar:at'room temperature, the ristocetin A moves 15 cm. when the system issubjected'to 140 volts and 1;5=2.0 for 22hours across the electrodesplaced in the potassium chloride solution. The said strips are incubatedwith- Bacillus subtilis. Under" identical electrophoretic conditionsristocetin B trave1s7'.5 cm; A'clean'separationof ri'st'ocetin B andristoetin "A; isrthus obtained wherimiired' in'a singlesolution andsubjected to thefoi'egoing-paper electrophoreses; 1

' Titration ofthe antibiotic shows the presence of -both weakly acidicand basic groups. Titration with acid gives an equivalent weightof'ab'out 12001400 with a midpointnear pH 7.0. Meas'urements'of thefreezing point depressions in aqueous solutions indicate. a molecu larweight of about '2500 and ultracentrifugation indicates a molecularweight of about 4000. The antibiotic ristocetin A is-very stable atneutral and acidic pHs and can be boiled in water at pH 2.5 for 30minutes without destroying significant-1y the antibiotic activitythereof.

Crystalline ristocetin A sulfate obtained in-the above manner is solublein water, dilute acids, and dilute alkalis; but itis generally insolubleinmost organic solvents including methyl alcohol, ethyl alcohol, ether,dioxane, ch: roform, and glacial-acetic acid.

Broth dilutionassays -using;:several strains of streptococci organismsto determine the inhibitory concentratiohs of ristocetin-B andristocetin-A show 'that'the activity ratio of'ristocetin B to-ristocetinA- is-betweenabout 3 -to 4l Thus, the'results'of'the brothdilutionas'say' studies indicate that crystalline ristocetin B has a potency ofbetween about 3000 to 4000-units-per-mg.-

with the standard crystalline ristocet-in-A potency-0E 1000 'P r t Theantibiotic ristocetin -A--l1as been therapeutically efiective onintravenous administration inhumansagainst certain pathogenicorganisms-eausing'pneumonia, -acute bronchitis, 'andenterococcalabacteremim 'Ihehntibiotic ristocefin- A has been found tobe bactericidalagainst many organisms, including pneumococci,streptococci, staphylococci, clostridia, corynebacteria,andniycobacteri-a, and many of the microorganisms against which it iseffective; including staphlocoecig donot readily develop strainresistance toj'the o f the bactericidalactionz- Q g Others may readilyadapt the invention for use under conditions of'servicegbyemployingioneor moi-cot the'novei featnres disclosedor'equivlentsthereof. As at p'resenfadvisdwith"respectto the'apparent scope of ourinvention, we desire to'claim' thefoll'oviing subject matter.

We claim: p I 1. 5 method for the"preparation of non toxic,acidadtiitien sjalts or ristceetin A which comprisesthe steps" of (a rying'a filteredrennentation beer'resinting-n-om the deepcultureaerobrefernientationcf the rnicr'oorganism Nocardia luridq with an-activate'd carbon-'to adsorb tnereon the ristocetijiaetivity presentiinth filtered beer, (5) 'filterirrg the slurry to-"separate flieearbcn'and washing the carbon repeatedly with an acidified, a ueo s; polar,organic solventto fr'ee the adsoibed ristocetin acid-addition saltstherefronzi, '(c) ririovi the organicsolvent from the c'oriibiii''washings" fesult'ihg aqueous' soldt'ioir to about 5,0; d); 'iiassiiigocetin acid-addition salts hie a'ckedi acomdadiusting' tie pet or the""aitionsnts seine 7 5 volume of ristocetin A sulfate,

en ages 1-2 r (a). was ing t e ca bo dat 1 I'iO extia 'q .i PSO a unaflo m et (f) thereafter washing tne carban acidified,

aqueous, polar, organic solventand collec g n eluate from the colpngp,andjg) co'rfianthating thefractions having anRf"value"of"betwenabout-0:40td0.60 to obtain the ristocetin Aacid-addition salt "asa-solid residue. M V

' 2; A method fortlie' preparation of "non toxie, crystalline,acid-addition salts "of which comprises the steps of (d) slurnying afiltered fermentationbeer resulting from the deep cultureaerobicfermentation of the microorganism'Nuurdialtifiizdwith an activatedcarbon to adsorb thereon the ristocetin activity'piesent in the filteredbeer, (b) filtering the slurry-to separate the carbon and washing thecarbon repeatedly with acidified, aqueous, polar, organic solventto"free*the adsorbed ristocetin acid-addition salts thei'frohi,-(c)removing the organic solvent from the combined washings andadjusting thepH of the resulting aqueous sohitionto'about' 510; (11) passing; theaqueous solution of ristocetin acid additionsalts through a carbon"chromatographic column packed with a composition comprisin'g'sixgramseach of carbon and diatomaceoiis earth-per of; solids present insaid aqueous solution to adsorb the -ristocetin acid-addition, salts onthe carbon, (e) washing'the carboncolumn with water to removeeirt'raneous'; water-soluble, unadsorbed material, (7) ther'ea'fterwashing the carbon column with an acidified, aqueous,-"po1ar, organicsolvent and collecting fractions ot e1nate-nom tnece1ne n (g)concentrating the fractions having an Rf value of between about 0.40 to060 until they '-co1itain 5 0%"by weight vonne-er iist'dctin A acidaddition salt, (12) passing the concentrate through an alunnna colunmprepared by slurrying alumina ih watef, adjusting the pH of the slurry:to 3.0, washing 'the' treated alumina successively with water and analkanol and"'air=drying, (i) washing the alumina columnhavingadsorbedtneie on T the ris tocetin A 'aid addition salt'withfapolaig' organic solvent to rein-emerging impurities, (j) freeing theadsorbed ristocetin A acid-addition salt from'the'falumina by washingthe alumina repeatcdly'with a solution containing equal parts byvoluiiibfi-watrhhd a -polar, organic solvent, (k) concentrating andlyophilizing' the collected washings to obtain an amorphous,- solid;ristoc'etin-A '-aeid=addition" saltand '(l) crystallizirig the solidsalt from an. aqueous alcohol solution.

3. A method for the preparationof crystalline-ristocetin A sulfate whichcomprises thev steps of (a)-slurrying a filtered fermentation beerresulting from the deep culture aerobic fermentation of themicroorganism Nocardia lurida with an activated carbon to adsofb thereonthe ristocetinactivity present in the 'filtred "beer, (b) filtering wereseparatetheca'rbon and washing the carbonrepeatdly' with 'ai'i'aqueou'sacetone. mixture containing 40% by volume of acetone and tour,milliliters of six normal sulfuric acid per'liter of aqueous acetonemixture to free the adsorbed ristocetinsulfates therefrom, (c) removingthe acetone 'the'combined and '-adjustin g tlie pI-I- of the resultingaqueous solutionto about 5.0, -(d) passing the aqueous solution ofristoee'tin sulfates through a-rcarbon' chromatographic column packedmthae'ompositien' comprising six grams each of carbon anddiatomaceons-earth per gram of solids present in said aqueoussolu'tion--tov adsor-b-the -1'istocetin sulfates on the carbon, (e) washing thecarbon column with water to remove extraneous, water-soluble'unad sorbedmaterial, (f) thereafter washing the carbon columnwithanfaquecusacetonemiirture containing 40% by volume ofacetone andoi'len'iillilite'f of six normal sulfuric acid per 'liti ofaqneonsaeetene iriixture and collecting fractions of eluate'fi'ofnthecolum (g') concentratin'g"the A fb'etween about 0.40 to: 0.60lm'fll tne eentnasoz y weight (h) passing tlifioilcentrate through analumina column prepared by slurrying alumina in water, adjusting the pH{of the slurry to 3.0, washing the treated alumina successively withwater and methanol and air drying, (i) washing the alumina column havingadsorbed thereon the ristocetin A sulfate with absolute methanol toremove organic impurities, (j) freeing the adsorbed ristocetin A suliatefrom the alumina by washing the alumina repeatedly with a mixturecontaining equal parts by volume of water and methanol, (k)concentrating and lyophilizing the collected washings to obtain theamorphous, solid, ristocetin A sulfate and (I) crystallizing the solidsulfate from an aqueous ethanol s s e 14 References Cited in the file ofthis patent Philip et 8.1.2 Antibiotics Ann., 1956-57, pp. 699-705, Oct.17-19, 1956.

Goldstein et al.: German app. SNA20,923I Va/30h, Bekauntgemacht am 1,December 1955, klasse 30h, gruppe 6 (8 pp. spec.).

Waksman et aL: The Actinomycetes and Their Antibiotics, pub. 1953, pp.134, 135, 144, 145, 153, 173,

10 180, 202 and 21s.

Absts. of Paper Nos. 91 thru 94, presented at the Antibiotic SymWashington, DC, Oct, 17-19, 1956 (4 pp.).

1. A METHOD FOR THE PREPARATION OF NON-TOXIC, ACIDADDITION SALTS OFRISTOCETIN A WHICH COMPRISES THE STEPS OF (A) SLURRYING A FILTEREDFERMENTATION BEER RESULTING FROM THE DEEP CULTURE AEROBIC FERMENTATIONOF THE MICROOGANISM NOCARDIA LURIDA WITH AN ACTIVATED CARBON TO ADSORBTHEREON THE RISTOCETIN ACTIVITY PRESENT IN THE FILTERED BEER, (B)FILTERING THE SLURRY TO SEPARATE THE CARBON AND WASHING THE CARBONREPEATEDLY WITH AN ACIDIFIED, AQUEOUS, POLAR, ORGANIC SOLVENT TO FREETHE ADSORBED RISTOCETIN ACID-ADDITION SALTS THEREFROM, (C) REMOVING THEORGANIC SOLVENT FROM THE COMBINED WASHINGS AND ADJUSTING THE PH OF THERESULTING AQUEOUS SOLUTION TO ABOUT 5.0, (D) PASSING THE AQUEOUSSOLUTION OF RISTOCETIN ACID-ADDITION SALTS THROUGH A CARBONCHROMATOGRAPHIC COLUMN PACKED WITH A COMPOSITION COMPRISING SIX GRAMSEACH OF CARBON AND DIATOMACEOUS EARTH PER GRAM OF SOLIDS PRESENT IN SAIDAQUEOUS SOLUTION TO ADSORB THE RISTOCETIN ACID-ADDITION SALTS ON THECARBON, (E) WASHING THE CARBON COLUMN WITH WATER TO REMOVE EXTRANEOUS,WATER-SOLUBLE, UNADSORBED MATERIAL, (F) THEREAFTER WASHING THE CARBONCOLUMN WITH AN ACIDIFIED, AQUEOUS, POLAR, ORGANIC SOLVENT AND COLLECTINGFRACTIONS OF ELUATE FROM THE COLUMN, AND (G) CONCENTRATING THE FRACTIONSHAVING AN RF VALUE OF BETWEEN ABOUT 0.40 TO 0.60 TO OBTAIN THERISTOCETIN A ACID-ADDITION SALT AS A SOLID RESIDUE.